1. Field of the Invention
This invention relates to a thermostable double strand-specific nucleic acid degrading enzyme (nuclease) (duplex-specific nuclease: to be referred to as DSN hereinafter) and a gene of said enzyme. It also relates to a novel thermostable DSN which can be easily produced using a recombinant protein expression technique, and a method for producing said DSN. It also relates to a novel thermostable DSN derived from an organism belonging to Brachyura, more illustratively derived from an organism belonging to Majidae, further illustratively derived from the genus Chionoecetes. It further relates to a method for digesting nucleic acid using said DSN and a method for detecting RNA using said DSN, and a reagent kit to be used in the aforementioned methods.
2. Brief Description of the Background Art
Nucleases which show DNase (DNA degrading enzyme) activity have so far been found in the digestive gland or hepatopancreas of some crustaceans, particularly Decapoda such as shrimps, crabs and the like. Particularly, it has been revealed that a king crab liver-derived nuclease is a characteristic enzyme which shows a DNase activity highly specific to double-stranded nucleic acid, and this enzyme has been called as duplex-specific nuclease (DSN).
i) Solenocera melantho (a Mud Shrimp) DNase
A nuclease derived from Decapoda such as shrimps, crabs and the like was firstly purified from Solenocera melantho of the family Solenoceridae as a hepatopancreas-derived DNase (Biochim Biophys Acta, 1036, 95-100(1990)). The Solenocera melantho DNase had a molecular weight (about 44 kDa) which was larger than that of a bovine hepatopancreas-derived DNase. It was confirmed that the Solenocera melantho DNase does not have a sugar chain, and it was considered that its large molecular weight is due to its long polypeptide chain. Although the Solenocera melantho DNase had a metal ion requirement and optimum activity pH, which were close to those of a bovine pancreas-derived DNase, it did not show an RNase (RNA degrading enzyme) activity. In addition, it was shown that the Solenocera melantho DNase has a resistance to trypsin digestion. From a result of the analysis of its amino acid composition, it was shown that the Solenocera melantho DNase is highly bridged intramolecularly through 18 disulfide bonds by 36 Cys residues.
ii) Penaeus japonicus (Kuruma Shrimp) DNase
A nuclease having similar molecular weight has also been purified from the hepatopancreas of Penaeus japonicus of the family Penaeidae (Biochim Biophys Acta, 1209, 209-214(1994)), and its cDNA sequence has been revealed (Biochem J, 346 Pt 3, 799-804(2000)). In addition to its DNase activity, the Penaeus japonicus nuclease showed a low level of RNase activity. Based on the homology of amino acid sequences, it was shown that the Penaeus japonicus nuclease belongs to the family of DNA/RNA nonspecific endonuclease (DRNSN) typified by the Serratia marcescens nuclease, rather than the bovine DNA degrading enzyme I-like protein (bovine DNase-I-like protein). Amino acid sequence of the Penaeus japonicus nuclease has 402 amino acid residues and consists of a mature enzyme of 381 residues and a presumed signal peptide of 21 residues. The Penaeus japonicus nuclease has 11 Cys residues, and 10 Cys residues among them formed 5 intramolecular disulfide bonds, and the remaining one Cys residue was linked to a thiol compound having an estimated molecular weight of from 500 to 700 Da. Wang et al. have succeeded in expressing a cloned nuclease gene in Escherichia coli, but the expressed protein did not show the nuclease activity (Biochem J, 346 Pt 3, 799-804(2000)).
iii) Paralithodes camtschaticus (King Crab) DSN
A nuclease having similar molecular weight has also been purified from the hepatopancreas of Paralithodes camtschaticus of the family Lithodidae, Anomala (.58,681-691(1993) (Biokhimia, 58,681-691(1993)), and its cDNA sequence has been revealed (Genome Res,12,1935-1942(2002)). Amino acid sequence of the Paralithodes camtschaticus nuclease has 407 amino acid residues and consists of a mature enzyme of 380 residues and a presumed signal peptide of 27 residues, and has a homology of 64% with the Penaeus japonicus nuclease.
Although the nuclease sequence of Paralithodes camtschaticus had also an NUC domain which is common to the DRNSN, as a result of characterization of the purified enzyme, to our surprise, the Paralithodes camtschaticus nuclease showed strong digestion selectivity for a double-stranded DNA substrate and hardly showed its activity for a single-stranded DNA. Also, the Paralithodes camtschaticus nuclease hardly showed digestion activity for an RNA substrate, and in the case of a DNA-RNA hybrid double strand, efficiently digested its DNA molecule alone. In addition, it hardly showed digestion activity for a short double-stranded DNA containing a mismatch. Thus, characteristic substrate specificity of the Paralithodes camtschaticus nuclease was revealed, and this enzyme was called “duplex-specific nuclease” (DSN) (Genome Res, 12, 1935-1942(2002), BMC Biochem, 9, 14(2008)).
Although Shagin et al. have cloned a cDNA of Paralithodes camtschaticus DSN and expressed its mature Paralithodes camtschaticus DSN, prepared by removing a presumed signal peptide, in Escherichia coli as an N-terminal His-tag fusion protein, the recombinant protein did not have the enzyme activity (Genome Res, 12, 1935-1942(2002)). Anisimova et al. have succeeded in purifying a soluble recombinant Paralithodes camtschaticus DSN having enzyme activity from an inclusion body in which the recombinant DSN molecule was aggregated in Escherichia coli, through a series of procedures including denaturation, refolding and activation (Biochemistry (Mosc),71,513-519(2006)). Additionally, the inventors of the present invention have previously isolated a Paralithodes camtschaticus DSN gene, and carried out expression of a recombinant Paralithodes camtschaticus DSN from said DSN gene having a signal peptide sequence, using a baculovirus-insect cell expression system. As a result, the inventors of the present invention have succeeded in expressing and purifying a soluble recombinant Paralithodes camtschaticus DSN which does not require refolding, and the thus obtained recombinant DSN showed a duplex-specific nuclease activity which was similar to that of the natural origin DSN (Expression of duplex-specific nuclease derived from Paralithodes camtschaticus by insect cells-baculovirus system. Eleventh Annual Meeting of Japanese Society for Marine Biotechnology P4-1(May 24 to May 25 in 2008)).
iv) Pandalus borealis (a Red Shrimp) DSN
It has been shown that a nuclease purified from the digestive gland of Pandalus borealis of the family Pondalidae, Caridea also has digestion selectivity for a double-stranded DNA substrate and hardly degrades single-stranded DNA (WO99/07887). In contrast to the Paralithodes camtschaticus DSN, this Pandalus borealis DSN is a heat-labile enzyme which has an optimum activity temperature of 25° C. and is inactivated by a heating at 70° C. for 30 minutes or at 94° C. for 2 minutes. As an application of the Pandalus borealis DSN making use of this heat-labile nature, a method for removing carry over contamination of PCR products has been disclosed (WO99/07887), although amino acid sequence and cDNA sequence of the Pandalus borealis DSN are not disclosed.
v) Other DSN Homologues
DSN homologues having homology with the amino acid sequence of Paralithodes camtschaticus DSN have been found in some species of Decapoda. Molthathong et al. have isolated a cDNA encoding for a DSN homologue from the hepatopancreas of Penaeus monodon (black tiger shrimp) of the family Penaeidae. Its estimated amino acid sequence was possessed of 89% of homology with the sequence of Penaeus japonicus (Fish Shellfish Immunol, 22, 617-627(2007)). As other DSN homologous sequences, a mRNA sequence derived from Amphiuca crassioes (a red fiddler crab) of the family Ocypedidae [GenBank DQ862540] and a mRNA sequence derived from a Palaemonidae sp. (a species of the family Palaemonidae) [GenBank DQ862538] are found in the GenBank sequence data base. Classification of the Paralithodes camtschaticus DSN and other DSN homologues as novel nuclease family based on the evolutional genealogical tree analysis has been proposed (Gene, 418, 41-8(2008). Since there are no reports on these DSN homologue sequences regarding the isolation and enzyme activity of each protein, it is not yet confirmed whether or not these are translated actually as proteins having nuclease activities, whether or not the translation products have duplex-specific nucleic acid digestion activities such as of the Paralithodes camtschaticus DSN, and whether or not these have heat resistance.
In summary, those in which the duplex-specific nuclease activity was actually shown among the Decapoda-derived DSN, DSN-like nucleases and DSN homologues are only two species of the DSN of Paralithodes camtschaticus belonging to Anomura (Anomara) and DSN of Pandalus borealis belonging to Caridea. While the former has a heat resistance having an optimum activity temperature of around 60° C., the latter is a heat-labile enzyme having an optimum activity temperature of 25° C. A DSN which shows a duplex-specific nuclease activity and a DSN having heat resistance, which were derived from an organism belonging to Brachyura, for example derived from an organism belonging to the family Majidae, such as a genus Chionoecetes-derived, for example a Chionoecetes opilio (snow crab)-derived counterparts, have not so far been disclosed.
Although Other than the case of Decapoda, an enzyme which shows a duplex-specific nuclease activity has been found for example in an insect Culex quinquefasciatus (J Exp Biol, 209 (Pt 14), 2651-9(2006)), heat resistance of said enzyme was not confirmed and its expression is limited to the salivary gland of female adults. Therefore, it seems difficult to industrially apply this enzyme by isolating it from the nature.
Namely, the nuclease derived from the hepatopancreas of Paralithodes camtschaticus was the only one heat-resistant DSN which is industrially applicable. Since the Paralithodes camtschaticus DSN has a characteristic selectivity for the digestion of nucleic acid molecules and it has heat resistance and shows its activity at high temperature, its various applications in the field of molecular biology have been shown. For example, SNP analysis (Genome Res, 12,1935-1942(2002), WO03/048378), normalization of cDNA library (Nucleic Acids Res, 32, e37(2004)), subtraction (Anal Biochem, 372,148-155(2008)) and analysis of telomere single strand overhand length (Nucleic Acids Res, 36, e14(2008)) and the like have been disclosed. However, the commercially available Paralithodes camtschaticus DSN enzyme reagents are purified products from the nature and therefore are considerably expensive. Although the recombinant Paralithodes camtschaticus DSN expressed in Escherichia coli is accumulated as an inactive inclusion body in the cell and it can be isolated as an active type enzyme after passing through solubilization, refolding and the like processes (Biochemistry (Mosc),71,513-519(2006)), the procedure requires multiple steps and therefore is complex.
Under such a background, a novel thermostable DSN enzyme has been in demand. Also, a novel thermostable DSN enzyme which can be easily produced making use of recombinant protein expression techniques has been in demand. Additionally, a novel thermostable DSN enzyme, derived from an organism belonging to Brachyura, for example derived from an organism of the family Majidae, for example derived from the genus Chionoecetes, has been in demand.